Isolation, characterization, and evaluation of three Citrus sinensis-derived constitutive gene promoters.

KEY MESSAGE: Regulatory sequences from the citrus constitutive genes cyclophilin (CsCYP), glyceraldehyde-3-phosphate dehydrogenase C2 (CsGAPC2), and elongation factor 1-alpha (CsEF1) were isolated, fused to the uidA gene, and qualitatively and quantitatively evaluated in transgenic sweet orange plants. The 5' upstream region of a gene (the promoter) is the most important component for the initiation and regulation of gene transcription of both native genes and transgenes in plants. The isolation and characterization of gene regulatory sequences are essential to the development of intragenic or cisgenic genetic manipulation strategies, which imply the use of genetic material from the same species or from closely related species. We describe herein the isolation and evaluation of the promoter sequence from three constitutively expressed citrus genes: cyclophilin (CsCYP), glyceraldehyde-3-phosphate dehydrogenase C2 (CsGAPC2), and elongation factor 1-alpha (CsEF1). The functionality of the promoters was confirmed by a histochemical GUS assay in leaves, stems, and roots of stably transformed citrus plants expressing the promoter-uidA construct. Lower uidA mRNA levels were detected when the transgene was under the control of citrus promoters as compared to the expression under the control of the CaMV35S promoter. The association of the uidA gene with the citrus-derived promoters resulted in mRNA levels of up to 60-41.8% of the value obtained with the construct containing CaMV35S driving the uidA gene. Moreover, a lower inter-individual variability in transgene expression was observed amongst the different transgenic lines, where gene constructs containing citrus-derived promoters were used. In silico analysis of the citrus-derived promoter sequences revealed that their activity may be controlled by several putative cis-regulatory elements. These citrus promoters will expand the availability of regulatory sequences for driving gene expression in citrus gene-modification programs.

Can chronic stretching change the muscle-tendon mechanical properties? A review.

It is recognized that stretching is an effective method to chronically increase the joint range of motion. However, the effects of stretching training on the muscle-tendon structural properties remain unclear. This systematic review with meta-analysis aimed to determine whether chronic stretching alter the muscle-tendon structural properties. Published papers regarding longitudinal stretching (static, dynamic and/or PNF) intervention (either randomized or not) in humans of any age and health status, with more than 2 weeks in duration and at least 2 sessions per week, were searched in PubMed, PEDro, ScienceDirect and ResearchGate databases. Structural or mechanical variables from joint (maximal tolerated passive torque or resistance to stretch) or muscle-tendon unit (muscle architecture, stiffness, extensibility, shear modulus, volume, thickness, cross-sectional area, and slack length) were extracted from those papers. A total of 26 studies were selected, with a duration ranging from 3 to 8 weeks, and an average total time under stretching of 1165 seconds per week. Small effects were seen for maximal tolerated passive torque, but trivial effects were seen for joint resistance to stretch, muscle architecture, muscle stiffness, and tendon stiffness. A large heterogeneity was seen for most of the variables. Stretching interventions with 3- to 8-week duration do not seem to change either the muscle or the tendon properties, although it increases the extensibility and tolerance to a greater tensile force. Adaptations to chronic stretching protocols shorter than 8 weeks seem to mostly occur at a sensory level.

Distinct beneficial effects of continuous vs accumulated exercise training on cardiovascular risk factors in Wistar rats.

We compared the effects of continuous exercise (CE) vs accumulated exercise (AE) training on CVD risk factors and heart of young male Wistar rats. The exercise training (ET) was performed in a swimming pool for 30-60 min/day, 5 days/week over 15 weeks. CE group performed the ET in a single long daily session (30-60 min), while AE group performed the ET at the same frequency, intensity, and duration of CE rats, but in three short bouts over the course of a day (10-20 min in three daily sessions). AE training was more efficient than CE in attenuating body and fat weight gain and inhibiting visceral adipocyte hypertrophy at the same food intake level. CE training was more efficient in improving systolic blood pressure, LDL/HDL cholesterol, and serum triglyceride. Both ET protocols increased heart function, decreased lipid peroxidation, and increased intracellular Hsp72 content in the heart. This work shows distinct beneficial effects of CE vs AE training suggesting that the prescription of one or other may be preferred to prevent the increase of a specific CVD risk factor.

Toluene Valence and Rydberg Excitations as Studied by ab initio Calculations and Vacuum Ultraviolet (VUV) Synchrotron Radiation.

The electronic spectroscopy of isolated toluene in the gas phase has been investigated using high-resolution photoabsorption spectroscopy in the 4.0-10.8 eV energy range, with absolute cross-section measurements derived. We present the first set of ab initio calculations (vertical energies and oscillator strengths), which we use in the assignment of valence and Rydberg transitions of the toluene molecule. The spectrum reveals several new features not previously reported in the literature, with particular relevance to 7.989 and 8.958 eV, which are here tentatively assigned to the π*(17a') ← σ(15a') and 1π*(10a″) ← 1π(14a') transitions, respectively. The measured absolute photoabsorption cross sections have been used to calculate the photolysis lifetime of toluene in the upper stratosphere (20-50 km).

Leaching behaviour and ecotoxicity evaluation of chars from the pyrolysis of forestry biomass and polymeric materials.

The main objective of this study was to assess the environmental risk of chars derived from the pyrolysis of mixtures of pine, plastics, and scrap tires, by studying their leaching potential and ecotoxicity. Relationships between chemical composition and ecotoxicity were established to identify contaminants responsible for toxicity. Since metallic contaminants were the focus of the present study, an EDTA washing step was applied to the chars to selectively remove metals that can be responsible for the observed toxicity. The results indicated that the introduction of biomass to the pyrolysis feedstock enhanced the acidity of chars and promote the mobilisation of inorganic compounds. Chars resulting from the pyrolysis of blends of pine and plastics did not produce ecotoxic eluates. A relationship between zinc concentrations in eluates and their ecotoxicity was found for chars obtained from mixtures with tires. A significant reduction in ecotoxicity was found when the chars were treated with EDTA, which was due to a significant reduction in zinc in chars after EDTA washing.

RFPID: development and 3D-printing of a female physical phantom for whole-body counter.

Whole-body counters (WBC) are used in internal dosimetry forin vivomonitoring in radiation protection. The calibration processes of a WBC set-up include the measurement of a physical phantom filled with a certificate radioactive source that usually is referred to a standard set of individuals determined by the International Commission on Radiological Protection (ICRP). The aim of this study was to develop an anthropomorphic and anthropometric female physical phantom for the calibration of the WBC systems. The reference female computational phantom of the ICRP, now called RFPID (Reference Female Phantom for Internal Dosimetry) was printed using PLA filament and with an empty interior. The goal is to use the RFPID to reduce the uncertainties associated within vivomonitoring system. The images which generated the phantom were manipulated using ImageJ®, Amide®, GIMP®and the 3D Slicer®software. RFPID was split into several parts and printed using a 3D printer in order to print the whole-body phantom. The newly printed physical phantom RFPID was successfully fabricated, and it is suitable to mimic human tissue, anatomically similar to a human body i.e., size, shape, material composition, and density.

Removal of phosphorus from wastewaters by biomass ashes.

Removal batch assays of phosphates from a synthetic wastewater (SWW) and a pulp and paper mill wastewater (PPWW) with two forestry biomass ashes were performed. The supernatants were not only chemically characterized but also the ecotoxicity was determined using two organisms: Vibrio fischeri and Artemia franciscana. The addition of fly ash and bottom ash to the SWW in solid/liquid (S/L) ratios of 3.35 and 9.05 g L(-1), respectively, achieved removal percentages of phosphates >97% for both ashes. The addition of fly ash and bottom ash to the PPWW in S/L ratios of 34.45 and 46.59 g L(-1), respectively, yield removal percentages of phosphates >90% for both ashes. According to the results of the Langmuir and Freundlich isotherms, the removal of phosphates from the SWW was mainly explained by surface removal mechanisms, while the removal from the PPWW was partially explained by multi-layer mechanisms. The supernatants resulting from the treatment of SWW and PPWW with both biomass ashes did not present acute ecotoxicity.

Analysis of eyewitness testimony using electroencephalogram signals.

Face recognition and related psychological phenomenon have been the subject of neurocognitive studies during last decades. More recently the problem of face identification is also addressed to test the possibility of finding markers on the electroencephalogram signals. To this end, this work presents an experimental study where Brain Computer Interface strategies were implemented to find features on the signals that could discriminate between culprit and innocent. The feature extraction block comprises time domain and frequency domain characteristics of single-trial signals. The classification block is based on a support vector machine and its performance for the best ranked features. The data analysis comprises the signals of a cohort of 28 participants.

New Radiation Dosimetry Estimates for [18F]FLT based on Voxelized Phantoms.

3'-Deoxy-3-[18F]fluorothymidine, or [18F]FLT, is a positron emission tomography (PET) tracer used in clinical studies for noninvasive assessment of proliferation activity in several types of cancer. Although the use of this PET tracer is expanding, to date, few studies concerning its dosimetry have been published. In this work, new [18F]FLT dosimetry estimates are determined for human and mice using Monte Carlo simulations. Modern voxelized male and female phantoms and [18F]FLT biokinetic data, both published by the ICRP, were used for simulations of human cases. For most human organs/tissues the absorbed doses were higher than those reported in ICRP Publication 128. An effective dose of 1.70E-02 mSv/MBq to the whole body was determined, which is 13.5% higher than the ICRP reference value. These new human dosimetry estimates obtained using more realistic human phantoms represent an advance in the knowledge of [18F]FLT dosimetry. In addition, mice biokinetic data were obtained experimentally. These data and a previously developed voxelized mouse phantom were used for simulations of animal cases. Concerning animal dosimetry, absorbed doses for organs/tissues ranged from 4.47 ± 0.75 to 155.74 ± 59.36 mGy/MBq. The obtained set of organ/tissue radiation doses for healthy Swiss mice is a useful tool for application in animal experiment design.

Behavioral changes in Japanese quails exposed to predicted environmentally relevant abamectin concentrations.

Abamectin (ABA) toxicity in fish, amphibian and mammals was already proven, but its effect on birds is almost unknown. Thus, the aim of our study is to assess the impact of exposure to water with ABA for 40 days at predicted environmentally relevant concentrations on the behavior of female Japanese quails (Coturnix coturnix japonica). The three following experimental groups (n = 10 each) were set: "control", quails exposed to drinking water, without ABA, "EC1x" and "EC1000x" (0.31 mg a.i./L and 310.0 a.i./L, respectively; via commercial formulation Kraft® 36EC). The open field test showed anxiolytic response in birds exposed to ABA. These birds did not show locomotor changes or aggressive behavior in the aggressiveness test. Quails exposed to the pesticide did not react to the introduction of an object in the experimental box during the object recognition test, and it suggested perception deficit due to ABA. Moreover, these birds did not recognize the cat (Felix catus) and the vocalization of a hawk (Rupornis magnirostris) as potential predatory threats. These responses also suggest anti-predatory behavior deficit due to the pesticide. Thus, our study is pioneer in showing that water with ABA, at tested concentrations, influences the behavior of C. coturnix japonica, as well as in highlighting the potential impacts of this pesticide on this group of birds.

Effect of heat stress and solar radiation on dry matter intake, biochemical indicators, production, and quality of Holstein and Jersey cows' milk / Efeito do estresse calórico e da insolação sobre consumo, indicadores bioquímicos, produção e qualidade do leite de vacas Holandês e Jersey

The objective of this work was to compare the dry matter intake, milk yield and quality, physiological and biochemical parameters in Holstein (n=10) and Jersey (n=10) cows under heat stress and insolation, in two treatments: CL - cooling by ventilation and sprinkling and HS - heat stress and insolation. Data were submitted to ANOVA. There was an interaction between treatment and breed and day effect for dry matter intake. For consumption in % of body weight, CL and Jersey cows consumed more. CL cows produced more milk and 3.5% fat-corrected milk. Feed efficiency was similar between treatments and breeds. Fat, lactose, total solids, and somatic cell score did not differ. The concentration of milk urea nitrogen was higher for CL cows. Milk from Holstein cows had greater stability to alcohol, and from HT cows had a greater freezing point of milk. HT cows had higher respiratory rates in the morning and surface temperatures in the afternoon. There were no differences in beta-hydroxybutyrate and glucose concentrations. Heat stress, with insulation, reduces intake, especially in Holstein cows, as well as milk production and increases the freezing point of milk, respiratory rate, and surface temperature.

O objetivo deste trabalho foi comparar o consumo de matéria seca, a produção e a qualidade do leite, os parâmetros fisiológicos e bioquímicos em vacas das raças Holandesa (n=10) e Jersey (n=10) sob estresse calórico e insolação, em dois tratamentos: CL - resfriamento por ventilação e aspersão; HS - estresse térmico e insolação. Os dados foram submetidos à análise de variância. Houve interação entre tratamento e raça e efeito de dia para consumo de matéria seca. Para consumo em % de peso vivo, vacas CL e Jersey consumiram mais. Vacas CL produziram mais leite e leite corrigido a 3,5% de gordura. A eficiência alimentar foi similar entre tratamentos e raças. Teores de gordura, lactose, sólidos totais e escore de células somáticas não diferiram. A concentração de nitrogênio ureico do leite foi maior para vacas CL. O leite das vacas Holandesas apresentou maior estabilidade ao álcool, e de vacas HT maior crioscopia. Vacas HT apresentaram maior frequência respiratória de manhã e temperatura superficial à tarde. Não houve diferenças para concentração de beta-hidroxibutirato e glicose. O estresse calórico, com insolação, reduz o consumo, especialmente em vacas Holandesas, bem como a produção de leite, com aumento da crioscopia, elevando a frequência respiratória e a temperatura superficial.

Chemical and ecotoxicological characterization of ashes obtained from sewage sludge combustion in a fluidised-bed reactor.

In 1999, the DEECA/INETI and the UBiA/FCT/UNL started a researching project on the partition of heavy metals during the combustion of stabilised sewage sludge (Biogran), in a fluidised-bed reactor, and on the quality of the bottom ashes and fly ashes produced. This project was entitled Bimetal and was funded by the Portuguese Foundation for Science and Technology. In this paper only the results on the combustion of Biogran are reported. The combustion process was performed in two different trials, in which different amounts of sewage sludge and time of combustion were applied. Several ash samples were collected from the bed (bottom ashes) and from two cyclones (first cyclone and second cyclone ashes). Sewage sludge, bed material (sand) and ash samples were submitted to the leaching process defined in the European leaching standard EN 12457-2. The eluates were characterized for a set of inorganic chemical species. The ecotoxicological levels of the eluates were determined for two biological indicators (Vibrio fischeri and Daphnia magna). The results were compared with the limit values of the CEMWE French Regulation. The samples were also ranked according to an index based on the chemical characterization of the eluates. It was observed an increase of the concentration of metals along the combustion system. The ashes trapped in the second cyclone, for both combustion trials, showed the highest concentration of metals in the eluates. Chemically, the ashes of the second cyclone were the most different ones. In the ecotoxicological point of view, the ecotoxicity levels of the eluates of the ashes, for both combustion cycles, did not follow the same pattern as observed for the chemical characterization. The ashes of the first cyclone showed the highest ecotoxicity levels for V. fischeri and D. magna. This difference on chemical and ecotoxicological results proves the need for performing both chemical and ecotoxicological characterizations of the sub-products of such type of thermal processes.

Resistance to Passion fruit woodiness virus in Transgenic Passionflower Expressing the Virus Coat Protein Gene.

We report the use of the coat protein (CP) gene from Passion fruit woodiness virus (PWV) to produce resistant transgenic plants of yellow passion fruit. A full-length CP gene from a severe PWV isolate from the state of São Paulo, Brazil (PWV-SP) was cloned into pCAMBIA 2300 binary vector, which was further introduced into Agrobacterium tumefaciens strain EHA 105. Leaf disks were used as explants for transformation assays, e.g., 2,700 and 2,730 disks excised from plants from the Brazilian cultivars IAC-275 and IAC-277, respectively. In vitro selection was performed in kanamycin. After transferring to the elongation medium, 119 and 109 plantlets of IAC-275 and IAC-277, respectively, were recovered. Integration of the PWV CP gene was confirmed in seven of eight plants evaluated by Southern blot analysis, showing different numbers of insertional events for the CP gene. Three transgenic plants (T3, T4, and T7) expressed the expected transcript, but the 32 kDa PWV CP was detected by Western blot in only two plants (T3 and T4). The results of three successive mechanical inoculations against the transgenic plants using three PWV isolates showed that the primary transformant T2 of IAC-277 was immune to all isolates.

Evolution of alternative methodologies of scorpion antivenoms production.

Scorpionism represents a serious public health problem resulting in the death of children and debilitated individuals. Scorpion sting treatment employs various strategies including the use of specific medicines such as antiserum, especially for patients with severe symptoms. In 1909 Charles Todd described the production of an antiserum against the venom of the scorpion Buthus quinquestriatus. Based on Todd's work, researchers worldwide began producing antiserum using the same approach i.e., immunization of horses with crude venom as antigen. Despite achieving satisfactory results using this approach, researchers in this field have developed alternative approaches for the production of scorpion antivenom serum. In this review, we describe the work published by experts in toxinology to the development of scorpion venom antiserum. Methods and results describing the use of specific antigens, detoxified venom or toxins, purified toxins and or venom fractions, native toxoids, recombinant toxins, synthetic peptides, monoclonal and recombinant antibodies, and alternative animal models are presented.

Ecotoxicological assessment of leachates from MSWI bottom ashes.

In this paper, chemical and ecotoxicological data of leachates from bottom ashes collected in different Municipal Solid Waste Incinerators (MSWI) are shown. The bottom ashes were collected in Belgium (three incinerators--samples B1 to B3), France, Germany, Italy and United Kingdom (one incinerator in each country--samples F1, D1, I1 and UK1, respectively). Both chemical and ecotoxicological characterizations of leachates were done on the framework of the European Directive 91/689/EEC and the European Council Decision 94/904/EC. This work was carried out under the European project called Valomat, which was supported by the European Commission through Brite-Euram III program. Twenty-one inorganic parameters were analyzed. The ecotoxicological assays were done under standard laboratory conditions, using the bacterium Photobacterium phosphoreum, the freshwater alga Pseudokirchneriella subcapitata, the crustacean Daphnia magna and the vegetable Lactuca sativa. Chemical data varied from sample to sample. Similar results were obtained in biological assays. The samples can be classified as ecotoxic/hazardous according to the French proposal for a Criterion and Evaluation Methods of Waste Ecotoxicity (CEMWE) and the German regulation on Hazardous Waste Classification (HWC). However, samples B1, B2, B3 and D1 comply the maximum limits for direct valorization category defined in the French Classification of Bottom Ashes based on their Polluting Potential (CBAPP). Sample B1 presented the lowest level of ecotoxicity, being considered as the most interesting to be used in the development of new materials for civil engineering works.

[Pulmonary lymphangioleiomyomatosis]. / Linfangioleiomiomatose pulmonar.

The authors present a clinical case of Lymphangioleiomyomatosis of the lung. They analyse the clinical, radiological and functional features and comment on the evolution and therapeutic approach of this rare disorder. To the authors, lung involvement as well as therapeutic decision are important features that affect the prognosis.

Molecular and functional characterization of metalloserrulases, new metalloproteases from the Tityus serrulatus venom gland.

Tityus serrulatus is a Brazilian scorpion species with great medical significance. While the effects of neurotoxins have been extensively studied, little is known about the proteases expressed in the venom gland of this arthropod. In this study, clones from a T. serrulatus (Ts) venom gland cDNA library were selected according to homology to proteases. The sequences were aligned in the database and classified by homology. Similarity and identity analyses of the sequences were carried out, and a phylogenetic tree was constructed with the sequences of other proteases. These cDNA sequences correspond to ten different metalloproteases, named metalloserrulases (TsMS). TsMS 1-9 belong to the metzincin family, which has three domains: signal peptide, propeptide, and metalloprotease domain; while TsMS 10 belongs to the gluzincin family. The proteolytic activity of the venom was inferred from the cleavage of fibrinogen, and the residues recognized by the proteases were determined by cleavage of a tripeptide library using a fluorescence resonance energy transfer assay. The Ts venom showed proteolytic activity on fibrinogen and preferential cleavage close to the basic residues K and R. Its activity could be inhibited by EDTA, indicating that the venom from this scorpion predominantly consists of metalloproteases.

ADP is a vasodilator component from Lasiodora sp. mygalomorph spider venom.

Members of the spider genus Lasiodora are widely distributed in Brazil, where they are commonly known as caranguejeiras. Lasiodora spider venom is slightly harmful to humans. The bite of this spider causes local pain, edema and erythema. However, Lasiodora sp. spider venom may be a source of important pharmacological tools. Our research group has described previously that Lasiodora sp. venom produces bradycardia in the isolated rat heart. In the present work, we sought to evaluate the vascular effect of Lasiodora sp. venom and to isolate the vasoactive compounds from the venom. The results showed that Lasiodora spider venom induced a concentration-dependent vasodilation in rat aortic rings, which was dependent on the presence of a functional endothelium and abolished by the nitric oxide synthase (NOS) inhibitor L-NAME. Western blot experiments revealed that the venom also increased endothelial NOS function by increasing phosphorylation of the Ser¹¹77 residue. Assay-directed fractionation isolated a vasoactive fraction from Lasiodora sp. venom. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) assays identified a mixture of two compounds: adenosine diphosphate (ADP, approximately 90%) and adenosine monophosphate (AMP, approximately 10%). The vasodilator effects of Lasiodora sp. whole venom, as well as ADP, were significantly inhibited by suramin, which is a purinergic P2-receptor antagonist. Therefore, the results of the present work indicate that ADP is a main vasodilator component of Lasiodora sp. spider venom.

Characterization of chars produced in the co-pyrolysis of different wastes: decontamination study.

The present work is devoted to the study of the decontamination of chars obtained in the co-pyrolysis of plastics, biomass and tyre wastes. The chars were extracted with several organic solvents of different polarities either individually or in sequence. The ability of each selected extractant to remove toxic pollutants was evaluated by comparing the extraction yields and by characterizing the crude extracts with a combination of chemical analysis and toxicity bioassays. Also, the mineral composition of the treated and non-treated chars was assessed. The results obtained in this study indicate that hexane is the more efficient extraction solvent to be used in the organic decontamination of chars obtained in the co-pyrolysis of plastics, tyres and biomass. A sequential extraction with solvents of increasing polarity can provide a better decontamination of the raw pyrolysis char than any individual extraction. The compounds removed from the char during the decontamination process are mainly aliphatic hydrocarbons and aromatic hydrocarbons, therefore a material that may be upgraded to be used as a fuel and/or as raw material for the organic chemical industry.